The tyrosine protein kinase Lck is essential for signaling through the T-cell antigen receptor (TCR). Lck associates with the inner surface of the plasma membrane and its localization to membrane lipid rafts is thought to be necessary for TCR-induced signaling. Although Lck is preferentially localized to lipid rafts, a significant portion resides in the more disordered regions of the membrane. We wish to test the hypothesis that the raft excluded Lck functions. We propose to investigate whether Lck localized in the non-raft regions of the membrane can initiate TCR signaling, by characterizing the differentially localized Lck and downstream targets and investigating the mechanistic basis for their localization. Using mutant versions of Lck and structural analogs of palmitic acid and farnesol designed to either target Lck, LAT (critical adaptor protein) and Ras (a key member of the Ras Erk Map kinase pathway) to lipid rafts or prevent their entry into rafts, we propose to determine the effect of the intramembrane localization of Lck, LAT and Ras on TCR-induced signaling. Although Lck's role as the initiator of TCR-signaling is well established, we have evidence that Lck is needed at a second step in the T-cell signaling cascade to regulate the kinase activity of c-Raf-1. Studies are proposed to characterize the interaction between Lck and c-Raf-1, including their co-localization in cells. Although regulation of the activity of Lck by tyrosine phosphorylation is well characterized, Lck is predominantly serine phosphorylated. We propose to characterize the serine phosphorylation of Lck and investigate its influence on the function and three dimensional structure of Lck.